ABSTRACT
An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.
Subject(s)
Fluorescein/chemistry , Fluorescent Dyes/chemical synthesis , Type C Phospholipases/metabolism , Fluorescein/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Molecular Structure , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase CABSTRACT
The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-delta1 in a continuous assay.
Subject(s)
Fluorescein/chemical synthesis , Fluorescent Dyes/chemical synthesis , Type C Phospholipases/analysis , Models, Chemical , Time FactorsABSTRACT
The susceptibility of the taiga tick Ixodes persulcatus Schulze to 15 pyrethroids has been evaluated. The knockdown time after exposure of ticks to pyrethroids tested was quite similar. It was revealed that the lethal doses of type I pyrethroids (permethrin-like) induced the rapid attachment of ticks to host. In contrast, the fatal poisoning with type II pyrethroids (alpha-CN-pyrethroids) made the ticks unable to attach completely. Possible mechanisms of the acceleration and the prevention of attachment are discussed.
